Type 1 diabetes accounts for about 10% of all cases of diabetes and its incidence continues to increase worldwide. While newer treatment strategies have facilitated improved outcomes, the long-term microvascular complications remain the major cause of morbidity and mortality in patients with type 1 diabetes. Cardiovascular diabetic autonomic neuropathy (DAN) has disabling clinical consequences and may facilitate malignant arrhythmias by altering electrical stability and impairing myocardial blood flow. There is a recognized need for novel mechanism-based biomarkers that accurately predict pathways activated in the complication-prone individual with DAN. This proposal is in response to a request of application (RFA-DK-06-004) and directly addresses this critical gap in our diagnostic capability. The overall goals of this proposal are to develop and validate novel biomarkers using a mass spectrometry (MS) based approach followed by a microarray-based proteomic platform combined with MS to elucidate pathways that contribute to type 1 DAN. A major focus of the proposal will involve development of a microarray-based proteomic platform that can readily be transferred to high throughput clinical applications. Using highly sensitive and specific gas chromatography MS we have recently characterized and validated specific markers of oxidative stress in plasma of humans with CAD and diabetes. These markers serve as molecular fingerprints for specific oxidation pathways. We have also developed a microarray based proteomic platform that can accurately identify and screen modified proteins from the plasma proteome. We predict that using our MS approach we will be able to identify differential oxidation of plasma proteins which uniquely identifies subsets of patients vulnerable for diabetic damage. We hypothesize that novel oxidatively modified protein biomarkers will predict phenotypic expression of DAN in diabetic patients and clinical responses to redox-modulating therapy in this patient subgroup. The specific aims of this proposal are: Aim 1: To determine oxidative biomarkers that predict presence of DAN in subjects with type 1 diabetes. Aim 2: To define oxidative biomarker profiles in a cohort of patients with established DAN treated with combination antioxidant therapy or placebo. Aim 3: To determine if alterations in biomarker profile are predictive of functional measures of DAN. Collectively, these studies using novel techniques and a systematic approach will provide insights into the pathogenesis of DAN and identify novel biomarkers for risk stratification, laying the groundwork for future large scale prospective studies.